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Supplemental Materials and Methods
Cell cultures. Mouse vascular smooth muscle cells (VSMCs) were obtained from aortae of 8 to 10
weeks old C57BL6 mice (Charles River Breeding Laboratories, Inc. Wilmington, MA). Cells were
isolated, cultured, and characterized as previously described [Castellot JJJ, Favreau LV, Karnovsky
MJ, Rosenberg RD. Inhibition of vascular smooth muscle cell growth by endothelial cell-derived
heparin. Possible role of a platelet endoglycosidase. J Biol Chem. 1982; 257: 11256-11260].
Identification of cultured cells as VSMCs was performed by indirect immunofluorescence staining for
the smooth muscle specific -actin (clone 1A4, Sigma). Cells were used for mineralization studies at
passages 8 through 12.
Aortic ring cultures. Renal arteries were collected from the freshly sacrificed Wistar-Furth rats.
Each artery was dissected further into three 2 to 3mm aortic rings, which were cultured under
serum-free conditions in medium with 3.8 mM concentration of PO43- as previously described
[Lomashvili K, Garg P, O'Neill WC. (2006). Chemical and hormonal determinants of vascular
calcification in vitro. Kidney Int. 2006; 69: 1464-1470]. After 9 days of culture with or without purified
TG2 (5 g/ml) added to the medium, arterial rings were analyzed for Ca2+ incorporation using the ocresolphthalein complexone Liquicolor kit (Stanbio Labs).
Real-time RT-PCR. Total RNA was isolated from the cultured mouse VSMCs with the Trizol
reagent (Invitrogen), and further purified with the RNAeasy kit (Qiagen). First-strand cDNA was
synthesized from 1 g of total RNA by using SuperScript III reverse transcriptase (Invitrogen). Realtime RT-PCR was performed by using the QuantiTect SYBR Green PCR kit (Qiagen). Reactions
were performed in triplicates for the each pair of primers, using the ABI 5700 Sequence Detector.
Results were analyzed by using the Microsoft Excel program.
Activation of -catenin pathway. The ß-catenin-dependent TCF/LEF transcriptional luciferase
reporter (TOP-FLASH) and the control reporter construct with the mutated TCF binding sites (FOPFLASH) were obtained from Upstate Biotechnology. The VSMC or COS-1 cells were plated at
5x104 /well in the 24-well plates and allowed to attach overnight. Either TOP-FLASH or FOPFLASH plasmid was co-transfected with -galactosidase plasmid at 10:1 ratio using the Fugene
reagent according to the manufacturer's instructions (Roche). At 24 h after transfection, cell cultures
were supplemented with purified guinea pig liver transglutaminase 2 (5g/ml) (Sigma) or purified
recombinant Wnt3a (10 ng/ml) (R&D), and cultured for 48 hours. Cells were lysed in the passive
lysis buffer and assayed for luciferase activity (Promega) according to the manufacturer's
-galactosidase activity, measured using specific substrate (Promega), was used
as the reference in order to normalize the luciferase activity for the transfection efficiency. The
average activities and standard deviations were derived from the triplicate transfected samples.
Proliferation assay. Cell proliferation was measured by the colorimetric assay with the MTS
tetrasolium compound (Owen’s reagent) (Promega). Cells were synchronized by overnight serum
starvation, cultured for 72 hours in 5% serum with or without purified TG2, and cell proliferation was
measured following the manufacturer’s protocol. All experiments were performed in triplicates.
Microarray analysis. Poly(A)+ RNA was selectively amplified from 0.5 – 1 µg of total RNA samples
with the BD SMART cDNA Synthesis kit and the BD Advantage 2 PCR Enzyme System (Clontech).
Approximately 4µg cDNA samples from three independent VSMC cultures were labeled with the
ULYSIS Alexa Fluor 546 (Invitrogen/Molecular Probes). Mouse 32K oligonucleotide microarray set
(Qiagen-Operon) was printed on aminosylane coated slides at Tufts Core facility. Labeled samples
were hybridized with microarrays in 1x hybridization buffer (Amersham) with 20% formamide for 44
hrs at 42°C. Slides were scanned on the ScanArray 4000 scanner (Packard Bioscience) and
analyzed with the ImaGene software (BioDiscovery). Statistical analyses were performed with the
package limma from the BioConductor project (www.bioconductor.org). ¾ Quartile was calculated
for all negative control spots on every array; and maximal value among all arrays was selected as
the threshold. Genes were selected as expressed if their expression value exceeded the threshold
on at least 3 arrays.
Statistical analysis of the results was performed using the Microsoft Excel software. Values are
expressed as mean+/- SD. Student’s unpaired two-tailed test was employed. Values of p<0.05 were
considered significant. All data shown have been confirmed in at least one independent experiment.
Supplemental Figure 1: Exogenous TG2 enhances ex vivo vascular calcification in rat renal
arteries.
Phosphate – medium supplemented with PO43- to the final concentration of 3.8 mM,
Phosphate+TG2 - medium supplemented with PO43- (3.8 mM) and purified guinea pig TG2 (Sigma)
(5 g/ml). Ca2+ levels were normalized to the total protein (determined using Bradford assay). Data
were obtained by analysis of four sets of arterial rings, derived from two animals.